According to the NHLBI, airway diseases are the leading cause of U.S. deaths due to lung disease. Defects in epithelial homeostasis and repair underlie pathologic remodeling seen in many lung disorders and contribute to chronic inflammation, fibrosis and airway obstruction. The overarching rationale of this proposal is that novel therapeutic strategies to prevent or reverse airway pathology and to engineer lung tissue from pluripotent stem cells will be greatly enhanced by a more thorough understanding of endogenous human regional epithelial stem cells, how they self renew, produce committed progenitors and differentiate to create a functional polarized epithelium. The team combines expertise in cell and developmental biology, stem cell biology and human lung pathobiology and exploits frog, mouse and human models in three highly interrelated aims. Aim 1 builds on a comprehensive next generation sequencing database of dynamic changes in mRNAs and microRNAs (miRs) in human bronchial epithelial cells undergoing differentiation in vitro. It tests the hypothesis that specific miRs regulate progenitor cell proliferation and cell fate decisions in vivo during generation and repair of a mucociliary epithelium. Aim 2 is based on studies in mice characterizing epithelial progenitor cells in large and small airways. It combines novel cell sorting strategies for cell isolation with 3D culture and transplantation techniques to characteriz poorly understood, but critically important, human small airway progenitors. Next generation RNA sequencing will then be used to generate a comprehensive database of mRNA and miRs in these cells. Aim 3 is based on studies showing dynamic regulation of grainyhead like transcription factors in an in vivo airway injury model and seeks to understand their role to integrate and coordinate epithelial polarity and barrier function during repair and regeneration. As well as bringing unique assays, model systems, technologies and resources to the Consortium, the proposed experiments will provide novel data about lung stem and progenitor cells that will help guide therapeutic strategies to mitigate or prevent airway pathology and to enhance lung repair, regeneration and reconstruction. RELEVANCE (See instructions): By identifying airway epithelial regional stem and progenitor cells and elucidating molecular mechanisms regulating their proliferation and differentiation, the studies address multiple outstanding questions central to lung repair and regeneration. The group brings unique skills and resources to the Consortium that will accelerate discovery of new therapies for lung diseases afflicting millions of people.